CN100569796C - Immunomodulatory peptides - Google Patents

Abstract

The invention discloses the peptide with SEQ ID NOs 1～24, described Toplink induces person monocytic cell or neutrophil to produce super-oxide; Can induce intracellular Ca2+ increase in human peripheral blood mononuclear cell or the neutrophil; Can combine with formyl peptide receptor or class formyl peptide receptor 1; Can induce the external chemotactic migration of person monocytic cell or neutrophil; Can induce the flailing action in formyl peptide receptor express cell or class formyl peptide receptor 1 express cell; Can be through the activation of formyl peptide receptor or class formyl peptide receptor 1, thus the irritation cell external signal is regulated the protein kinase phosphorylation; Or can stimulate the Akt phosphorylation through the activation of formyl peptide receptor or class formyl peptide receptor 1.

Description

Immunomodulatory peptides

Background of invention

Invention field

The present invention relates to immunomodulatory peptides.The formyl peptide receptor family of expressing in phagocytic cell such as neutrophil and monocyte (formyl peptide receptor (FPR) and class formyl peptide receptor 1 (FPRL1)) plays an important role in the host defense pathogenic infection (1,2).Known this receptor and Toxins, pertussis-responsive Gi albumen coupling (1,2).The activation-inducing G β γ subunit of FPR dissociates from G α i subunit, and β γ--and subunit is regulated Phospholipase C β or the kinase whose activation of lipositol 3-(1,2).The downstream signal conduction of the activation-inducing complexity of these effector molecules causes the various kinds of cell response, as the generation of chemotactic migration, threshing and super-oxide.

Description of Related Art

Most of full agonists are induced the cell signal of many complexity, cause the immune response of final complexity.In these immune responses, many reactions be the host cell pathogenic agent of removing invasion suitable function essential substantially, but some reactions are side effects unnecessary in the immune response.In the drug development field, the side effect that reduces or eliminates drug candidate has become the focus of research.For realizing this goal, the selective immune response conditioning agent or the selective antagonist (3,4) of special receptor attempted to develop by several method by many study group.

From endogenous or synthetic approach, identified multiple FPR agonist (1,2).Comprise bacterial peptide class (N-formyl radical-methionyl-leucyl-phenylalanine (fMLF)), HIV-coating structural domain (T20 and T21), and the agonist of host source property (AnnexinI and A β ₄₂) (5-7).Before this, inventor of the present invention has reported a kind of synthetic peptide part, Trp-Lys-Tyr-Met-Val-D-Met-NH ₂(hereinafter), can stimulate white corpuscle, as monocyte and neutrophil (8-11) with " WKYMVm " expression.Le etc. confirm that WKYMVm can combine (12) with formyl peptide receptor (FPR) and class formyl peptide receptor 1 (FPRL1).Because WKYMVm a kind ofly has the small peptide of high affinity to the wide spectrum acceptor, so it can be used as the useful matter of the signal conduction of research FPR-or FPRL1-mediation.But, to advancing the selectivity immunomodulator or the selective antagonist of shape at special receptor, and the research of the screening of molecular diversity, only form by micromolecular compound, therefore very limited, so also need to continue the identification new compound.

Summary of the invention

First target of the present invention provides novel immunomodulatory peptides, and its aminoacid sequence is selected from SEQID NO:1～SEQ ID NO:24; Or derived from the material of peptide, the aminoacid sequence of described peptide is selected from SEQ ID NO:1～SEQ ID NO:24.

Second target provides a kind of pharmaceutical composition, comprises that aminoacid sequence is selected from the peptide of SEQ ID NO:1～SEQ ID NO:24; Or derived from the material of peptide, the aminoacid sequence of described peptide is selected from SEQID NO:1～SEQ ID NO:24.

The 3rd target provides a kind of method that is changed the disease (condition) of following or causing by leukocyte count or active state for the treatment of, described method comprises that the aminoacid sequence of the host's administering therapeutic significant quantity that gives required treatment is selected from the peptide of SEQ ID NO:1～SEQ ID NO:24, or is selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence.

The 4th target provides a kind of host's of increasing quantity of leucocyte or strengthens the method for leukocytic active state, this method comprises the host that needs greater amount white corpuscle or the stronger leukocyte activation state peptide with the treatment significant quantity, this peptide ammino acid sequence is selected from SEQ ID NO:1～SEQ ID NO:24, or is selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence.

The 5th target is to provide a kind of method that extracellular Ca2 increases in the white corpuscle of inducing for the such patient who treats of needs, this method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQID NO:1～SEQ ID NO:24, or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The 6th target is the method that super-oxide generation in a kind of person monocytic cell of inducing or the neutrophil is provided for the patient of this treatment of needs, this method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ ID NO:24, or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The 7th target is the method that a kind of human peripheral blood mononuclear cell's of inducing chemotactic migration is provided for the patient of this treatment of needs, this method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ ID NO:24, or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The 8th target is that the patient for this treatment of needs provides a kind of method of inducing threshing reaction in formyl peptide receptor express cell or class formyl peptide receptor 1 express cell; this method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ ID NO:24; or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The 9th target is that the patient for this treatment of needs provides a kind of method that combines the peptide of formyl peptide receptor on formyl peptide receptor express cell or class formyl peptide receptor 1 express cell or class formyl peptide receptor 1 respectively with the WKYMVm competition; this method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ ID NO:24; or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The tenth target is the method that the patient for this treatment of needs provides extracellular signal adjusting protein kinases in a kind of stimulation formyl peptide receptor express cell or class formyl peptide receptor 1 express cell or formyl peptides (peptiin); this method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ ID NO:24; or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The 11 target is to provide a kind of method that stimulates Akt in formyl peptide receptor express cell or class formyl peptide receptor 1 express cell for the such patient who treats of needs; this method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ ID NO:24; or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The tenth two objects provides a kind of isolating Nucleotide, and described nucleotide coding aminoacid sequence is selected from the peptide of SEQ ID NO:1～SEQ ID NO:24.

The 13 target provides a kind of carrier that comprises separating nucleotide, and described nucleotide coding aminoacid sequence is selected from the peptide of SEQ ID NO:1～SEQ ID NO:24.

Its its feature of the present invention and advantage are in conjunction with hereinafter description and accompanying drawing can find out significantly that wherein similar in the accompanying drawings reference character is represented identical or similar part.

The accompanying drawing summary

Accompanying drawing is incorporated into and constitutes the part of specification sheets, for example understands embodiment of the present invention, and with specification sheets, explains principle of the present invention jointly.

Fig. 1 shows the [Ca in the RBL-2H3 cell (B) that RBL-2H3 cell (A) that WKYMVm, peptide of the present invention (WKGMVm, WKRMVm, WKYMVE, WKYMVR) or fMLF express FPR-or FPRL1-express ²⁺] _iEffect;

Fig. 2 shows that WKYMVm, peptide of the present invention (WKGMVm, WKRMVm, WKYMVE, WKYMVR) and fMLF are to being attached on FPR or the FPRL1 ¹²⁵The WKYMVm displacement of 1-mark;

Fig. 3 shows WKYMVm, peptide of the present invention (WKGMVm, WKRMVm, WKYMVE, WKYMVR), fMLF and wkymvm, in the RBL-2H3 cell that FPR-or FPRL1-are expressed the ERK phosphorylation influence;

Fig. 4 shows WKYMVm, peptide of the present invention (WKGMVm, WKRMVm, WKYMVE, WKYMVR), fMLF and wkymvm, in the RBL-2H3 cell that FPR-or FPRL1-are expressed the Akt phosphorylation influence;

Fig. 5 shows that WKYMVm increases the cell exocrine of the RBL-2H3 cell that stimulates FPR-or FPRL1-expression by intracellular Ca2+;

Fig. 6 shows WKYMVm, peptide of the present invention (WKGMVm, WKRMVm, WKYMVE, WKYMVR), fMLF and wkymvm, to the cell exocrine effect of N-formyl radical-methionyl-leucyl-phenylalanine (fMLF), to peptide-inductive [Ca ²⁺] influence that increases.

Fig. 7 shows the activity of WKYMVm by P13K and MEK, stimulates the chemotactic migration of the RBL-2H3 cell of FPR-or FPRL1-expression; And

Fig. 8 shows that WKYMVm, peptide of the present invention (WKGMVm, WKRMVm, WKYMVE, WKYMVR), fMLF and wkymvm are to chemotactic influence.

Detailed description of the preferred embodiments

Formyl peptide receptor (FPR) and class formyl peptide receptor 1 (FPRL1) play a significant role in immune response.The invention provides peptide class derived from WKYMVm.Many peptides can stimulate FPR or FPRL1 to cause the increase of calcium, but peptide of the present invention, as WKGMVm, WKRMVm and 6 ^ThD-Met alternate peptide only makes the calcium in the cell that FPRL1-expresses increase, and the calcium in the cell that FPR-expresses is increased.Use ¹²⁵The competitive assay of I-WKYMVm shows that many peptides not only make the calcium in the cell that FPR-expresses increase, and WKGMVm, WKRMVm and 6 ^ThD-Met alternate peptide also can with ¹²⁵The I-WKYMVm competition is in conjunction with FPR.Different with the calcium increase of Phospholipase C-adjusting, WKGMVm, WKRMVm and 6 ^ThD-Met alternate peptide can stimulate extracellular regulated protein kinase (ERK) and the Akt activation in the cell that FPR-expresses.The functional outcome of WKYMVm is that this peptide passes through Ca ²⁺With the ERK path, can stimulate the flailing action in the FPR cell and the chemotaxis of cell respectively.The peptide class is as WKGMVm, WKRMVm and 6 ^ThD-Met alternate peptide just by inducing the chemotactic migration to stimulate the FPR cell, does not still have flailing action.In general, the present invention has confirmed that for the first time as important chemoattractant chemical attractants acceptor, FPR can be carried out difference by different peptide parts and regulate according to part-special mode.

First preferred embodiment according to the present invention, peptide of the present invention comprise the aminoacid sequence that is selected from SEQ ID NO:1～SEQ ID NO:24 or derived from the material of SEQ ID NO:1～SEQ ID NO:24 peptide.SEQ ID NO:1～SEQ ID NO:24 is as described below:

Trp-Lys-Gly-Met-Val-D-Met-NH ₂ (WKGMVm；SEQ ID NO：1)，

Trp-Lys-Tyr-Met-Gly-D-Met-NH ₂ (WKYMGm；SEQ ID NO：2)，

Trp-Lys-Tyr-Met-Val-Gly-NH ₂ (WKYMVG；SEQ ID NO：3)，

Trp-Arg-Tyr-Met-Val-D-Met-NH ₂ (WRYMVm；SEQ ID NO：4)，

Trp-Glu-Tyr-Met-Val-D-Met-NH ₂ (WEYMVm；SEQ ID NO：5)，

Trp-His-Tyr-Met-Val-D-Met-NH ₂ (WHYMVm；SEQ ID NO：6)，

Trp-Asp-Tyr-Met-Val-D-Met-NH ₂ (WDYMVm；SEQ ID NO：7)，

Trp-Lys-His-Met-Val-D-Met-NH ₂ (WKHMVm；SEQ ID NO：8)，

Trp-Lys-Glu-Met-Val-D-Met-NH ₂ (WKEMVm；SEQ ID NO：9)，

Trp-Lys-Trp-Met-Val-D-Met-NH ₂ (WKWMVm；SEQ ID NO：10)，

Trp-Lys-Arg-Met-Val-D-Met-NH ₂ (WKRMVm；SEQ ID NO：11)，

Trp-Lys-Asp-Met-Val-D-Met-NH ₂ (WKDMVm；SEQ ID NO：12)，

Trp-Lys-Phe-Met-Val-D-Met-NH ₂ (WKFMVm；SEQ ID NO：13)，

Trp-Lys-Tyr-Met-Tyr-D-Met-NH ₂ (WKYMYm；SEQ ID NO：14)，

Trp-Lys-Tyr-Met-(Phe/Trp)-D-Met-NH ₂ (WKYM(F/W)m；SEQ ID NO：15)，

Trp-Lys-Tyr-Met-Val-Glu-NH ₂ (WKYMVE；SEQ ID NO：16)，

Trp-Lys-Tyr-Met-Val-Val-NH ₂ (WKYMW；SEQ ID NO：17)，

Trp-Lys-Tyr-Met-Val-Arg-NH ₂ (WKYMVR；SEQ ID NO：18)，

Trp-Lys-Tyr-Met-Val-Trp-NH ₂ (WKYMVW；SEQ ID NO：19)，

Trp-Lys-Tyr-Met-Val-NH ₂ (WKYMV；SEQ ID NO：20)，

Lys-Tyr-Met-Val-D-Met-NH ₂ (KYMVm；SEQ ID NO：21)，

Lys-Tyr-Met-Val-NH ₂ (KYMV；SEQ ID NO：22)，

Tyr-Met-Val-D-Met-NH ₂ (YMVm；SEQ ID NO：23)，

And Met-Val-D-Met-NH ₂(MVm; SEQ ID NO:24).

The peptide class of SEQ ID NO:1～SEQ ID NO:24 with separate and basically purified form exist.

Described peptide comprises chooses quilt-NH on carboxyl wantonly ₂The amino-acid residue that replaces.

Peptide of the present invention has a kind of following characteristic at least:

(a) induce the generation of super-oxide by person monocytic cell or neutrophil;

(b) increase by human peripheral blood mononuclear cell or neutrophil inductive intracellular Ca2+;

(c) combine with formyl peptide receptor or class formyl peptide receptor 1;

(d) chemotactic of external evoked person monocytic cell or neutrophil migration;

(e) induce the cell of formyl peptide receptor expression or the flailing action of the cell that class formyl peptide receptor 1 is expressed;

(f) by activating formyl peptide receptor or class formyl peptide receptor 1 irritation cell external signal-adjusting protein kinase phosphorylation; And

(g) by activating formyl peptide receptor or class formyl peptide receptor 1 stimulation Akt phosphorylation.

Second preferred embodiment according to the present invention, the invention provides a kind of pharmaceutical composition, comprise that aminoacid sequence is selected from the peptide of SEQ ID NO:1～SEQ ID NO:24, or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence.

Comprise as the peptide of activeconstituents or the described composition of material, can comprise surpassing a kind of medicinal diluent, be selected from salt solution, buffering salt, glucose, water, glycerine or ethanol, and thinner is not limited to these.

Composition can have different application according to disease and dosage.The actual amount that should be appreciated that activeconstituents should consider that correlative factor decides, comprise disease, the patient symptom of treatment severity, with other medicines applied in any combination (for example, chemotherapeutics), age, sex, patient's body weight, diet, administration time, the route of administration of selection and the ratio of composition.Even dosage and approach are adjusted according to the type and the severity of disease, every day, composition still can single dose or be divided into dosed administration 1-3 time.

The composition that comprises peptide of the present invention or material can oral administration or parenteral administration.Parenteral admin is meant through other administration except that mouth, comprises per rectum, vein, intraperitoneal and muscle, intra-arterial, skin, nasal cavity, suction, through eye and subcutaneous importing administration.

The pharmaceutical preparation that comprises peptide or material can be made any form, as oral dosage form, injection solution or topical formulation.Pharmaceutical dosage form is preferably made oral and form (true solution, suspensoid or emulsion) and most preferred oral form the injectable administration, as tablet, capsule, soft capsule, aqua, pill, particle etc.

In formulation preparation, peptide can directly add soft capsule and without any excipient, or mixes with carrier or dilute and make suitable formulation afterwards.The example of suitable carriers has starch, water, salt solution, Ringer's solution, glucose etc.

The 3rd preferred embodiment according to the present invention provides treatment quantity of leucocyte or active state to change the method for the disease of following or causing.Described method comprises the host to the treatment of this kind of needs, and the aminoacid sequence of administering therapeutic significant quantity is selected from the peptide of SEQ ID NO:1～SEQ ID NO:24 or is selected from the material of SEQ ID NO:1～SEQ ID NO:24 preface peptide derived from aminoacid sequence.Disease condition can be bacterium, mycoplasma, yeast, fungi, virus infection or inflammatory reaction.

The 4th preferred embodiment according to the present invention provides a kind of leukocyte increasing quantity or has improved the method for white corpuscle active condition.Described method comprises that to needs greater amount or the leukocytic host's administration of stronger activity the aminoacid sequence of administering therapeutic significant quantity is selected from the peptide of SEQ ID NO:1～SEQ ID NO:24 or is selected from the material of SEQ ID NO:1～SEQ ID NO:24 preface peptide derived from aminoacid sequence.

The 5th preferred embodiment according to the present invention, the patient who treats like this for needs provides a kind of method that extracellular Ca2 increases in the white corpuscle of inducing.This method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ ID NO:24, or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The 6th preferred embodiment according to the present invention is for the patient of this treatment of needs provides the method that super-oxide produces in a kind of person monocytic cell of inducing or the neutrophil.This method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ ID NO:24, or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The 7th preferred embodiment according to the present invention provides a kind of method of the human peripheral blood mononuclear cell's of inducing chemotactic migration for the patient of this treatment of needs.This method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ ID NO:24, or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The 8th preferred embodiment according to the present invention is for the patient of this treatment of needs provides a kind of method of inducing threshing reaction in formyl peptide receptor express cell or class formyl peptide receptor 1 express cell.This method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ IDNO:24, or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The 9th preferred embodiment according to the present invention is for the patient of this treatment of needs provides a kind of method that combines the peptide of formyl peptide receptor on formyl peptide receptor express cell or class formyl peptide receptor 1 express cell or class formyl peptide receptor 1 respectively with the WKYMVm competition.This method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ ID NO:24, or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The tenth preferred embodiment according to the present invention is for the patient of this treatment of needs provides signal adjusting protein kinase whose method in extracellular in a kind of stimulation formyl peptide receptor express cell or class formyl peptide receptor 1 express cell.This method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ IDNO:1～SEQ ID NO:24, or be selected from the material of SEQ ID NO:1～SEQID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

The 11 preferred embodiment according to the present invention; the patient who treats like this for needs provides a kind of method that stimulates Akt in formyl peptide receptor express cell or class formyl peptide receptor 1 express cell; this method comprises to described patient uses the peptide that a certain amount of aminoacid sequence is selected from SEQ ID NO:1～SEQ IDNO:24; or be selected from the material of SEQ ID NO:1～SEQ ID NO:24 peptide derived from aminoacid sequence, institute's consumption for to induce or the treatment of desensitization or prevention effectively.

In above-mentioned the 3rd to 11 preferred embodiment, host who is treated or patient may be subjected to owing to infect the invasion, particularly cytomegalovirus infection of associated diseases, rheumatoid arthritis, blue Mu Shi sacroiliitis, gout, sepsis syndrome, hyperpyrexia, ulcerative colitis, enterocolitis, osteoporosis, periodontal disease, glomerulonephritis, the inflammatory reaction of lung chronic noninfective, sarcoidosis, smoker's lung, granuloma forms, hepatic fibrosis, pulmonary fibrosis, graft-rejection, graft is to host's disease, chronic myeloid leukemia, acute myeloid leukemia, neoplastic disease, bronchial asthma, I type insulin-dependent diabetes mellitus, arteriosclerosis, atherosclerosis, psoriasis, chronic bone-marrow-derived lymphocyte leukemia, common variable immunodeficiency, disseminated inravascular coagulation, systemic scleroderma, encephalomyelitis, pulmonary inflammatory reaction, high IgE syndrome, cancer metastasis, growth of cancers, adoptive immunotherapy (adoptive immune therapy), acquired respiratory distress syndrome, Sepsis, pour into syndrome again, the post-operation inflammatory reaction, organ transplantation or alopecia.

The invention provides isolating Nucleotide, described nucleotide coding has the peptide that comprises SEQ ID NO:1～SEQID NO:24 aminoacid sequence.

The invention provides the carrier that comprises isolating Nucleotide, described nucleotide coding comprises the peptide of SEQ IDNO:1～SEQID NO:24 aminoacid sequence.

The invention provides and comprise the polypeptide that is selected from SEQ ID NO:1-SEQ ID NO:24 aminoacid sequence.

The present invention will explain in more detail with reference to the following example.But these examples are being gone up the restriction that all can not be interpreted as the scope of the invention in all senses.

Used material among the embodiment

Fmoc (protection) amino acid from Millipore (Bedford, MA).The Rapidamide resin available from Dupont (Boston, MA).Peripheral blood lymphocytes (PBMC) isolation medium (Histopaque-1077), cytochrome c and fMLF available from Sigma (St.Louis, MO).Fura-2 five acetoxyl group methyl esters (fura-2/AM) available from Molecular Probes (Eugene, OR).RPMI 1640 from Life Technologies (Grand Island, NY).Foetal calf serum of having dialysed and the bovine serum that replenishes available from Hyclone Laboratories Inc. (Logen, UT).PTX, GF109203X and PD98059 available from Calbiochem (San Diego, CA).LY294002 is available from BIOMOLresearch laboratories, and Inc. (Polymouth Meeting, PA).

With the synthetic peptide (8,9) of above-described solid phase method.In brief, peptide supports on the resin synthetic at rapidamide, and synthesizes according to the Fmoc/ tertiary butyl method of standard on acid-sensitive sense connection base.Determine the composition of peptide by aforementioned (8) amino acid analysis.

(13) according to the method described above carry out the cultivation of the RBL-2H3 and the RBL-2H3 cell that FPRL1-expresses of RBL-2H3, FPR-expression with the DMEM of the G418 that adds 20%FBS and 200g/ml.

Embodiment 1: neutrophil separates

(Ulsan Korea) gives by Ulsan Red Cross Blood Center to concentrate peripheral blood leucocyte.Human neutrophils is isolated in dextran precipitation, the cracking of red corpuscle hypo-osmoticity and the isolating standard method of lymphocyte gradient media according to above-mentioned (9).Isolating human neutrophils is used immediately.

Example 2: the influence that peptide produces super-oxide in the human neutrophils

Peptide WKYMVm, the peptide of SEQ ID NOs:1～24 and the activity that wkymvm produces super-oxide in the human neutrophils have been measured.(Winooski VT) measures the minimizing of cytochrome c, thereby quantizes the generation of superoxide anion for Bio-Tekinstruments, EL312e, and method is as describing in (14) by utilizing microtitration 96-orifice plate ELISA to read the plate device.(the 96-orifice plate contains 1x10 in every hole to human neutrophils ⁶RPMI 1640 substratum of cell/100 μ l) with 50 μ M cytochrome cs 37 ℃ of preincubation 1 minute, then with the peptide incubation of prescribed concentration.Carry out the measurement that super-oxide produces by the variation of measuring 550nm place's photoabsorption, 1 minute measuring space is once measured 5 minutes.At least carry out four independent experiments on each position and select activated amino acid peptide class.These results are presented in the Table I.

Stimulate neutrophil with the peptide of different concns, find that WKYMVm causes the generation of super-oxide in concentration dependence mode, during with the peptide of 100nM active maximum (data do not show).When some peptides, when producing as super-oxide in WRYMVm, WEYMVm, WKFMVm and the KYMVm irritation cell, many peptides produce super-oxide with the concentration of 100nM the time active a little less than.

Table I. the influence that peptide produces super-oxide in the human neutrophils ^a

Sequence b	O 2- (nmol/10 6Cell)	Sequence	O 2- (nmol/10 6Cell)
				WKYMVm-NH 2	37.3±6.94	WKFMVm-NH 2	37.3±3.56
WKGMVm-NH 2	14.2±3.42	WKYMYm-NH 2	25.8±3.89
				WKYMGm-NH 2	1.1±0.05	WKYM(F/W)m-NH 2	6.1±0.77
WKYMVG-NH 2	4.4±0.54	WKYMVE-NH 2	5.0±0.43
				WRYMVm-NH 2	47.1±11.23	WKYMVV-NH 2	5.0±0.21
WEYMVm-NH 2	52.9±12.78	WKYMVR-NH 2	16.3±1.57
				WHYMVm-NH 2	20.7±7.85	WKYMVW-NH 2	11.5±1.62
WDYMVm-NH 2	25.8±6.56	WKYMV-NH 2	9.2±0.55
				WKHMVm-NH 2	29.5±7.71	KYMVm-NH 2	36.0±3.56
WKEMVm-NH 2	4.1±0.21	KYMV-NH 2	7.5±0.61
				WKWMVm-NH 2	11.5±0.97	YMVm-NH 2	30.2±2.74

WKRMVm-NH 2	15.9±2.48	MVm-NH 2	0
				WKDMVm-NH 2	16.3±1.67	wkymvm-NH 2	0

^aMeasuring super-oxide by the minimizing of monitoring cytochrome c produces.

^bThe concentration of handling peptide is 100nM.

Also having measured concentration is the influence of the peptide of 10 μ M to the super-oxide generation.The result is presented in the Table II.Except that wkymvm, the activity that all peptides stimulate super-oxide to produce when 10 μ M concentration is the same with WKYMVm strong.Wherein WKWMVm, WKFMV and WKYMVW demonstrate the activity stronger than WKYMVm.

Table II. the influence that peptide produces super-oxide in the human neutrophils ^a

Sequence b	O 2- (nmol/10 6Cell)	Sequence	O 2- (nmol/10 6Cell)
				WKYMVm-NH 2	39.0±3.58	WKFMVm-NH 2	60.2±5.57
WKGMVm-NH 2	37.6±4.57	WKYMYm-NH 2	42.9±3.34
				WKYMGm-NH 2	27.3±2.61	WKYM(F/W)m-NH 2	45.6±7.76
WKYMVG-NH 2	27.3±1.40	WKYMVE-NH 2	33.6±6.43
				WRYMVm-NH 2	38.2±6.54	WKYMVV-NH 2	36.3±2.29
WEYMVm-NH 2	35.3±2.88	WKYMVR-NH 2	44.8±3.65
				WHYMVm-NH 2	20.7±7.85	WKYMVW-NH 2	56.8±4.60
WDYMVm-NH 2	37.3±5.66	WKYMV-NH 2	25.6±2.20
				WKHMVm-NH 2	39.3±4.10	KYMVm-NH 2	49.2±5.82
WKEMVm-NH 2	39.2±4.71	KYMV-NH 2	35.4±2.13
				WKWMVm-NH 2	65.0±12.10	YMVm-NH 2	41.8±3.46
WKRMVm-NH 2	41.7±8.32	MVm-NH 2	42.4±2.47
				WKDMVm-NH 2	37.3±2.78	wkymvm-NH 2	0

^aMeasuring super-oxide by the minimizing of monitoring cytochrome c produces.

^bThe concentration of handling peptide is 10 μ M.

Embodiment 3: peptide is to [Ca in the RBL-2H3 cell of FPR-or FPRL1-expression ²⁺] _iThe influence that increases

Peptide WKYMVm, sequence in the RBL-2H3 cell that FPR-expresses have been measured and are the peptide of SEQ IDNOs:1～24 and wkymvm to [Ca ²⁺] _iThe activity that increases.With every kind of RBL-2H3 cell that peptide stimulation FPR-expresses of 10 μ M, measure [Ca then ²⁺] _i[Ca ²⁺] _iThe level fluorescent method that utilizes Grynkiewicz measure (15) with fura-2/AM.In brief, the cell of preparation continues to shake with 3 μ Mfura-2/AM, 37 ℃ of incubations 50 minutes in the RPMI of fresh serum-free 1640 substratum.During each is analyzed, with 2x10 ⁶Cell is distributed to no Ca ²⁺Locke ' s solution (154mMNaCl, 5.6mM KCl, 1.2mM MgCl ₂, 5mM HEPES, pH 7.3,10mM glucose, and 0.2mM EGTA) in.Measurement is in the change in fluorescence of two excitation wavelength 340nm and 380nm and 500nm transmitted wave strong point, and the ratio of the fluorescence after will proofreading and correct is converted into [Ca ²⁺] _i[the Ca that monitoring increases ²⁺] _iThe peak level.The result is presented among Table III and Figure 1A.Data are the representative of three independent experiments.

Table III. the influence that peptide increases intracellular Ca2+ in the RBL-2H3 cell of FPR-expression ^a

Sequence	EC50(nM)	Sequence	EC50(nM)
				WKYMVm-NH 2	47.4±10.94	WKFMVm-NH 2	17.7±4.79
WKGMVm-NH 2	Non-activity	WKYMYm-NH 2	665.4±81.53
				WKYMGm-NH 2	Non-activity	WKYM(F/W)m-NH 2	57.3±10.30
WKYMVG-NH 2	Non-activity	WKYMVE-NH 2	Non-activity
				WRYMVm-NH 2	54.9±8.33	WKYMVV-NH 2	Non-activity
WEYMVm-NH 2	317.4±29.33	WKYMVR-NH 2	Non-activity
				WHYMVm-NH 2	31.7±3.36	WKYMVW-NH 2	Non-activity
WDYMVm-NH 2	98.9±17.51	WKYMV-NH 2	Non-activity
				WKHMVm-NH 2	279.8±35.86	KYMVm-NH 2	384.8±33.13
WKEMVm-NH 2	1332.8±88.75	KYMV-NH 2	Non-activity
				WKWMVm-NH 2	18.8±5.31	YMVm-NH 2	569.1±63.38
WKRMVm-NH 2	Non-activity	MVm-NH 2	Non-activity
				WKDMVm-NH 2	1329.5±207.20	wkymvm-NH 2	Non-activity

^aFrom load the increase of cell monitoring intracellular Ca2+ of fura-2.

In the RBL-2H3 cell that FPR-expresses, the WKYMVm peptide is induced [Ca in the mode that concentration relies on ²⁺] _iIncrease, maximum activated concentration is about 300nM (data not shown).WKYMVm is to [Ca in the FPR cell ²⁺] _iThe active EC50 of increase be 47nM (Table III).In peptide of the present invention, although WHYMVm, WKWMVm and WKFMVm compare with peptide WKYMVm, demonstrate the avidity higher to FPR, the activity that other peptide demonstrates is not as WKYMVm (Table III).Especially, WKGMVm, WKYMGm and 6 ^ThD-Met metathetical peptide does not produce [Ca ²⁺] _iIncrease, express RBL-2H up to handling FPRL1-with the concentration of 20 μ M ₃Cell (Table III and Figure 1A).The peptide of N-end and the brachymemma of C-end is to [the Ca among the FPR ²⁺] _iIncrease also non-activity (Table III).These results show Tyr ³And D-Met ⁶To [Ca ²⁺] _iFPR in the increase activates most important.

In the RBL-2H3 cell that FPRL1-expresses, peptide WKYMVm, sequence have been detected and are the peptide of SEQID NOs:1～24 and wkymvm to [Ca ²⁺] _iThe influence that increases.The RBL-2H3 cell that FPRL1-expresses stimulates with each peptide of 10 μ M, and has measured [Ca ²⁺] _i[Ca ²⁺] _iLevel measure with above-mentioned same method.Detect [Ca ²⁺] _iThe peak level.The result is presented among Table IV and Figure 1B.Data are the representative of three independent experiments.

Express in the RBL-2H3 cell at FPRL1-, the WKYMVm of 10 μ M concentration has demonstrated maximum activity (data not shown).WKYMVm is to [Ca in the FPRL1 cell ²⁺] _iIncreasing active EC50 is 0.6nM (Table IV).Be that all peptides are to [Ca in the FPRL1 cell with different in the FPR cell ²⁺] _iIncrease all be activated (Table IV).Some peptides demonstrate high-affinity (Table IV) as WRYMVm, WKWMVm, WKFMVm, WKYMYm and WKYM (F/W) M to FPRL1.In the FPR cell, do not influence [Ca ²⁺] _iWKGMVm, the WKYMGm and 6 that increase ^ThD-Met metathetical peptide also demonstrates [Ca in the FPRL1 cell ²⁺] _iThe activity that increases has the avidity lower to FPRL1 (Table IV and Figure 1B).The peptide of N-end and the brachymemma of C-end also stimulates [the Ca among the FPRL1 ²⁺] _iIncrease (Table IV).These results show, for the activation of FPR, and Tyr ³And D-Met ⁶To [Ca ²⁺] _iFPRL1 activated importance in the increase is less better.

Table IV. the influence that peptide increases middle intracellular Ca2+ in the RBL-2H3 cell of FPRL1-expression ^a

Sequence	EC50(nM)	Sequence	EC50(nM)
				WKYMVm-NH 2	0.60±0.090	WKFMVm-NH 2	0.23±0.042
WKGMVm-NH 2	21.32±2.104	WKYMYm-NH 2	0.29±0.061
				WKYMGm-NH 2	18.11±1.308	WKYM(F/W)m-NH 2	0.12±0.015
WKYMVG-NH 2	5945.8±176.100	WKYMVE-NH 2	502.87±64.965
				WRYMVm-NH 2	0.12±0.010	WKYMW-NH 2	1259.15±95.750
WEYMVm-NH 2	5.23±0.196	WKYMVR-NH 2	177.52±26.035

WHYMVm-NH 2	0.72±0.075	WKYMVW-NH 2	194.48±19.210
				WDYMVm-NH 2	14.28±1.225	WKYMV-NH 2	917.85±45.610
WKHMVm-NH 2	1.94±0.268	KYMVm-NH 2	3.01±0.232
				WKEMVm-NH 2	28.30±1.354	KYMV-NH 2	＞30000
WKWMVm-NH 2	0.16±0.027	YMVm-NH 2	17.15±0.889
				WKRMVm-NH 2	2.06±0.256	MVm-NH 2	＞30000
WKDMVm-NH 2	8.73±1.210	wkymvm-NH 2	Non-activity

^aFrom load the increase of cell monitoring intracellular Ca2+ of fura-2.

Embodiment 4: peptide to [ ¹²⁵I] WKYMVm and FPR or the influence of FPRL1 bonded

Based on the discovery that some peptides (WKGMVm, WKRMVm, D-Met6 substituted peptide) can not induce cytoplasmic calcium to increase, need further to judge whether peptide can combine with FPR.Detected peptide to being attached to FPR or FPRL1 ¹²⁵The displacement situation of I-mark WKYMVm.FPR-express the RBL-2H3 cell with [ ¹²⁵I] WKYMVm, do not having or having in the presence of the unmarked WKYMVm of incremental change or SEQ ID NOs:1～24 peptides incubation together.

The part binding analysis carries out (16) according to the modification method of aforementioned report.The WKYMVm of radioiodine mark ( ¹²⁵The I-mark) available from NEN Lifesciences (Boston, MA).In brief, FPR-or FPRL1-express RBL-2H ₃Cell is according to every hole 1X10 ⁵Cell is assigned in the 24-orifice plate, and overnight incubation., having or do not having in the presence of the 50 μ M unmark peptides after 2 hours with the blocking-up of blocking-up damping fluid (33mM HEPES, pH 7.5, the RPMI of 0.1%BSA) pair cell, with single concentration ¹²⁵The WKYMVm of I-mark is added in the cell with binding buffer liquid (PBS that contains 0.1%BSA), and under the sustained oscillation situation 4 ℃ of incubations 3 hours.Then sample is washed 5 times with ice-cold binding buffer liquid, and in every hole, add the lysis buffer (20mM Tris, pH 7.5,1%Triton X-100) of 200 μ l.After the room temperature cracking 20 minutes, collect lysate.Measure bonded with γ-line register ¹²⁵The radioactivity of I-mark WKYMVm.

The part binding analysis is the result be presented among Fig. 2.As shown in Figure 2, the mode that the WKYMVm of not only unlabelled WKYMVm but also WKGMVm, WKRMVm and D-Met6 substituted peptide relies on concentration, suppress [ ¹²⁵I] combination of WKYMVm.These results show, although the D-Met6-substituted peptide of WKGMVm, WKRMVm or WKYMVm can be incorporated into FPR, the cytoplasmic calcium that all peptides can not induce FPR-to express in the RBL cell increases.

Embodiment 5: peptide is expressed the influence of ERK phosphorylation in the RBL-2H3 cell to FPR or RPRL1

I) use the peptide irritation cell

The RBL-2H3 cell of cultivating is divided into 2X10 ⁶Cell, and stimulate with the WKYMVm that indicates concentration and peptide of the present invention and to specify the long time.FPR-or FPRL1-express the WKYMVm stimulation different time (Fig. 3 A) of RBL-2H3 cell with 1 μ M.Before the WKYMVm with 1 μ M handles, two kinds of cells carry out preincubation with following solution, GFX (15 minutes), 10 μ MBAPTA/AM (60 minutes) or the 50 μ MPD98059 (60 minutes) (Fig. 3 B) of LY294002 (15 minutes), the 5 μ M of PTX (24 hours), the 50 μ M of usefulness solvent (vehicle) or 100ng/ml.The RBL2H3 cell that FPR-or FPRL1-express stimulated 2 minutes or 5 minutes (Fig. 3 C) with the peptide of the present invention of 10 μ M respectively.

After the stimulation, with the RPMI washed cell of serum-free, and with lysis buffer (20mM Hepes, pH 7.2,10% glycerine, 150mM NaCl, 1%Triton X-100,50mM NaF, 1mMNa ₃VO ₄, 10 μ g/ml leupeptins, 10 μ g/ml aprotinins, and 1mM phenylmethylsulfonyl fluoride) and carry out cracking.Centrifugation washing agent insolubles (12,000Xg, 15 minutes, 4 ℃), and dissolved supernatant fraction shifted out, and store or use immediately at-80 ℃.Protein concn in the lysate is measured with Bradford protein analysis reagent.

Ii) electrophoresis and immunoblotting assay

Each sample (containing 30g protein) is carried out 10%SDS-PAGE separate, and phosphorylation ERK measures by immunoblotting assay with anti--phosphoric acid-ERK antibody.By utilizing concentrated sample buffer, carry out electrophoresis, the preparation protein example.The buffering system that part in the sample is described with Laemmli is then separated (17) by the 10%SDS-polyacrylamide gel.

Behind the electrophoresis, it is identical with conclusive evidence used sample size in experiment to carry out western blot analysis with anti--ERK2 antibody.Then with western blotting to nitrocellulose filter.Then by (Tris-buffer salt solution, 0.05%Tween-20) incubation seal nitrocellulose membrane with containing 5% skim-milk TBS.Subsequently, the anti--phosphoric acid-ERK antibody of film, anti--phosphoric acid-Akt antibody or anti--Akt antibody carry out incubation, wash with TBS.When carrying out PKC transposition (translocation) analysis, the antibody special with the PKC isozyme carries out incubation.Antigen-antibody complex develops the color by following manner, with the goat that is coupled to horseradish peroxidase of film and 1: 5000 dilution anti--rabbit igg or goat be anti--mouse IgG antibody carries out incubation, and carry out with enhanced chemiluminescence detection system.

Iii) result

Estimated in the RBL-2H3 cell that peptide expresses FPR-or FPRL1-, the influence of the cell transduction by FPR or RPRL1, the result shows in Fig. 3.The result of Fig. 3 is the representative of 3 independent experiments.

Since although some Toplink combine with FPR, they can not stimulate the calcium of PLC-mediation to increase, and have investigated them to the influence that other signal in some GPCRs downstreams conducts (ERKs and Akt), described other signal conduction is independent of PLC.Stimulate FPR-or FPRL1-to express the RBL-2H3 cell with 1 μ M WKYMVm, induce instantaneous ERKs to activate, show, occurred maximum activity (Fig. 3 A) respectively at 2 minutes or 5 minutes with after the peptide processing.When before stimulating with WKYMVm, with several inhibitor FPR-or FPRL1-expression RBL-2H3 cell are carried out pre-treatment, WKYMVm-induces ERKs to activate PTX and PD98059 sensitivity, shows that this incident is PTX-sensitive G-protein and is MEK-dependent (Fig. 3 B).The pre-treatment of P13K inhibitor (LY294002), pkc inhibitor (GF109203X or Ro-31-8220) or calcium chela and agent (BAPTA/AM) can not produce WKYMVm-inductive ERK and activate (Fig. 3 B).This shows that this incident is independent of P13K, Ca ²⁺Activate with PKC.Therefore it seems that WKYMVm induces [Ca by the independent signal pathway ²⁺] _iIncrease and the ERK activation.With anti--phosphoric acid-ERKs antibody has been investigated peptide of the present invention by western blot analysis to be influenced the ERK activated.Different with cytoplasmic calcium increase activity is that peptide (WKGMVm, WKRMVm, WKYMVR and WKYMVE) stimulates the ERKs phosphorylation (Fig. 3 C) in the FPR-expression RBL-2H3 cell.Should attentively be [the Ca that these peptides can not influence the PLC-mediation ²⁺] _iIncrease activity, this is an absorbing result.When FPRL1-expression RBL-2H3 cell stimulated with WKYMVm and peptide of the present invention, most of described peptides also caused the ERKs phosphorylation (Fig. 3 C) in the FPRL1 cell.This result is relevant with aforementioned result, and all Toplink stimulate the cytoplasmic calcium in the FPRL1-express cell to increase (Figure 1B).

Embodiment 6: peptide is expressed the influence of Akt phosphorylation in the RBL-2H3 cell to FPR-or RPRL1-

As everyone knows, activated chemoattractant acceptor is induced the activation (19) of Akt through P13K.Use the peptide irritation cell, carry out electrophoresis and immunoblotting assay according to method same among the embodiment 5.

FPR-or FPRL1-express the RBL-2H3 cell stimulates the different time (Fig. 4 A) with 1 μ M WKYMVm.Before handling with the WKYMVm of 1 μ M, two kinds of cells carry out preincubation (Fig. 4 B) with BAPTA/AM (60 minutes) or the 50 μ MPD98059 (60 minutes) of GFX (15 minutes), the 10 μ M of LY294002 (15 minutes), the 5 μ M of PTX (24hr), the 50 μ M of solvent or 100ng/ml.FPR-and FPRL1-expression RBL2H3 cell stimulated respectively 2 minutes or 5 minutes (Fig. 4 C) with WKYMVm and the peptide of the present invention of 10 μ M.Each sample (protein of 30 μ g) is carried out 10%SDS-PAGE, and anti--phosphoric acid-Akt antibody is measured to phosphorylation Akt by immunoblotting assay.By proving conclusively the sample that is used for testing same amount with anti--western blot analysis that Akt antibody carries out.The result is presented among Fig. 4.The result of Fig. 4 is the representative of 3 independent experiments.

Observe at FPR-and FPRL1-and express in the RBL-2H3 cell, it is time-dependent mode (Fig. 4 A) that WKYMVm stimulates inductive Akt phosphorylation.WKYMVm-induces the Akt phosphorylation to PTX, LY294002 sensitivity, but insensitive to GFX and BAPTA/AM, shows that this incident is the G-protein and the P13K-dependency (Fig. 4 B) of PTX-susceptibility.Not only FPR-expression RBL-2H3 cell is stimulated, can cause Akt phosphorylation (Fig. 4 C) with WKYMVm but also with peptide of the present invention (WKGMVm, WKRMVm, WKYMVE and WKYMVR).WKYMVm and peptide of the present invention also can stimulate the Akt phosphorylation (Fig. 4 C) in the FPRL1-expression RBL-2H3 cell.These results be associated with the ERKs phosphorylation of peptide in two kinds of cells (Fig. 3 C).As if because WKYMVm-inductive ERKs and Akt activate by the P13K activation and mediate, WKGMVm, WKRMVm, WKYMVE and WKYMVR successfully induce the signal conduction of the P13K-mediation in FPR downstream.

Embodiment 7: peptide is to the influence of exocytosis (exocytosis)

Granule secretion is one of mastocyte most important function (20).By (18) detection Hex secretion according to the method described above, investigate the influence of WKYMVm to granule secretion.In brief, express RBL-2H3 cell (2x105/well) overnight incubation in the tissue culturing plate of 24-hole of FPR or FPRL1.With cell Tyrode ' s damping fluid (137Mm NaCl, 12mM NaHO ₃, 5.6mM glucose, 2.7mM KCl, 1Mm CaCl ₂, 0.5mMMgCl ₂, 0.4Mm NaH ₂PO ₄, 0.1g/100ml BSA and 25mM HEPES, pH 7.4) wash, and stimulate with various peptides.WKYMVm with various concentration handles FPR-or FPRL1-expression RBL-2H ₃Cell (Fig. 5 A).Under the condition that has or do not have 10 μ MBAPTA/AM to exist, with WKYMVm stimulation two kinds of clones (Fig. 5 B) of 1 μ M.This response stimulus stopped by being placed on ice after 20 minutes.The β hexosaminidase that is secreted in the substratum is measured according to following method, with the 0.1M sodium citrate buffer solution (pH3.8) of 5mM p-nitrophenyl-N-ethanoyl-right-D-glycosamine of 50 μ l supernatant liquors or cell pyrolysis liquid and 25 μ l 37 ℃ of incubations 2 hours.When incubation finishes, add the Na of the 0.4M of 50 μ l ₂CO ₃Measure the absorbancy at 405nm place.The result is presented among Fig. 5.Data are the mean value ± S.E. of triple experiments.(mean value ± S.E.) is expressed as the per-cent of total Hex in the cell to numerical value.

Multiple concentration WKYMVm expresses the RBL-2H3 cell when stimulating to FPR-or FPRL1-, and it is (Fig. 5 A) that carries out in the mode that concentration relies on that caused Hex discharges.Express RBL-2H at FPR-or FPRL1- ₃In the cell, when stimulating, can demonstrate maximum activity (Fig. 5 A) respectively with 100nM or 10nM peptide.There has been report to point out that cytoplasmic calcium increases granule secretion among mastocyte such as the RBL-2H3 very crucial (18,20).Confirmed that also before stimulating with peptide, the intracellular Ca2+ chelating of handling by BAPTA/AM almost completely suppresses WKYMVm-inductive granule secretion (Fig. 5 B).

From these new discoveries as can be known, the substituted peptide of WKYMVm and many WKYMVm can induce cytoplasmic calcium to discharge, but some can not (WKGMVm, WKRMVm, D-Met6 substituted peptide), has investigated in the RBL cell these peptides to the influence of granule secretion.Handle FPR-(Fig. 6 A) or FPRL1-expression RBL-2H3 cell (Fig. 6 B) with each peptide of 10 μ M.Measure peptide-the induce secretion of Hex according to the method described above.Data are the mean value ± S.E. of triple experiments.

When stimulating the FPR cell with peptide (WKYMVm, WKGMVm, WKRMVm, WKYMVE, WKYMVR), can observe the granule secretion effect of some substituted peptides, but except WKGMVm-, WKRMVm-and the D-Mets-substituted peptide (Fig. 6 A).WKGMVm-, WKRMVm-and D-Met6-substituted peptide can not influence the granule secretion (Fig. 6 A) in the FPR-expression RBL-2H3 cell.These results are related fully with aforementioned result, and WKGMVm-, WKRMVm-and D-Met6-substituted peptide can not induce cytoplasmic calcium to discharge (Figure 1A).With different situation in the FPR-expression RBL-2H3 cell be, all peptides (WKYMVm, WKGMVm, WKRMVm, WKYMVE, WKYMVR), but do not comprise fMLF or wkymvm, stimulate the granule secretion (Fig. 6 B) in the FPRL1-expression RBL-2H3 cell.This result is also well related with aforementioned result, and the cytoplasmic calcium that peptide stimulates FPRL1-to express in the RBL-2H3 cell increases (Figure 1B).

Embodiment 8: the chemotactic influence of peptide pair cell

Carry out chemotaxis assay (analysis of improved Boyden chamber) (Neuroprobe Inc., Gaithersburg, MD) (18) with the porous chamber.In brief, polycarbonate filter (8 μ m aperture) wraps quilt in advance with HEPES-buffered RPMI 1640 substratum of the rat I collagen (Collaborative Biomedicals) of 50 μ g/ml.The filter that drying is wrapped quilt is placed in the 96-pore chamber that contains the different concns peptide.In RPMI, concentration is 1x10 with the RBL-2H3 cell suspension of expressing FPR or FPRL1 ⁶The serum-free RPMI of cell/ml, and the suspension of 25 μ l is positioned in the hole, upper strata of chemotaxis chamber, 96-hole.37 ℃ of incubations are after 4 hours, scrape off the not cell of migration, and the cell that will move strainer dewaters, fix, and the usefulness phenodin (Sigma, St.Louis MO) dye.The staining cell of 5 in the described hole high multiple visuals field of selecting at random (HPF) in (400X) counted.Fig. 7 A has shown the result of chemotaxis assay.Solvent, 50 μ M LY294002 (15 minutes) and the pretreated cell of 50 μ M PD98059 (60 minutes), the chemotaxis assay when carrying out 1 μ M WKYMVm processing, the result is presented among Fig. 7 B.The migrating cell number is measured by the cell in the counting high multiple visual field (400x).Data are the mean value ± SE of three independent experiments, and each experiment repeats twice.

Existing report, WKYMVm can be induced the chemotactic migration (11) of phagocytic cell such as monocyte and neutrophil.Le etc. confirmed by with FPR and FPRL1 bonded WKYMVm-inducing cell chemotaxis (12).Just as desired, to express in the RBL-2H3 cell at FPR-or FPRL1-, the chemotactic migratory activity that WKYMVm demonstrates has clock (bell-) type concentration dependent (Fig. 7 A).WKYMVm-inductive cell chemotaxis is to LY294002 and PD98059 sensitivity (Fig. 7 B).This result hints that WKYMVm-inductive cell chemotaxis is that P13K-and MEK-rely on.WKYMVm stimulates the chemotactic migration of the RBL-2H3 cell of FPR-or FPRL1-expression through P13K and MEK activity.

Measured peptide of the present invention and FPR-or FPRL1-have been expressed the influence of the cell chemotaxis of RBL-2H3 cell.The various peptides of multiple concentration are used for the chemotaxis assay of the RBL-2H3 cell of FPR-or FPRL1-expression.By counting, measure the migrating cell number at the high multiple visual field (400x) pair cell.Data are expressed as the mean value ± SE of two independent experiments, and each experiment repeats twice.

Express in the RBL-2H3 cell at FPR-, not only the chemotaxis (Fig. 8 A) of WKYMVm but also substituted peptide (WKGMVm, WKRMVm, WKYMVE and WKYMVR) inducing cell.The concentration of the needed substituted peptide of chemotaxis is higher than the concentration (Fig. 8 B) of WKYMVm.At relating to substituted peptide-induce the chemotaxis signal transduction path, checked the participation situation of PI-3 kinases and MEK-mediation signal conduction.When FPR-expresses the RBL cell with LY294002 or PD98059 pretreated the time, WKYMVm and substituted peptide-inductive RBL cell migration almost completely is suppressed (Fig. 7 B and data not shown).Express in the RBL-2H3 cell at FPRL1-, WKYMVm and peptide (WKGMVm, WKRMVm, WKYMVE and WKYMVR) inductive cell chemotaxis also demonstrates concentration dependent (Fig. 8 B).

In the present invention, confirmed that FPR can regulate with different parts for the first time, caused the cell signaling and the function result of difference.Regulate for the part-specificity that confirms FPR, prepared as multiple peptide listed in the Table I the strong active ligand of FPR.Wherein, WKGMVm-, WKRMVm-and D-Met6-substituted peptide can be incorporated into FPR, only induce the Akt of PI-3 kinases-mediation and the ERK of MEK-mediation to activate, and cause the chemotactic migration of cell.These peptides can not influence cytoplasmic calcium to be increased.Because peptide WKYMVm not only stimulates ERKs to activate, and the cytoplasmic calcium increase, cause chemotaxis and flailing action, mean that therefore FPR can carry out difference with different parts and regulate.

Recently, several pieces of reports have confirmed that some GPCRs can regulate (21,22) with different parts.In the part of GPCR-specificity is regulated, think that different parts induces the different conformational changes of acceptor, and induce acceptor and specific effector molecule or the proteic selectivity coupling of G-.In Table III and Figure 1A, confirmed to express in the RBL-2H3 cell at FPR-, WKGMVm, WKRMVm and D-Met6 substituted peptide can not stimulate PLC-mediation cytoplasmic calcium to increase.But these peptides stimulate ERKs and the Akt phosphorylation (Fig. 3 C) in the FPR-expression RBL-2H3 cell.In WKYMVm and peptide of the present invention-mediated cell signal conduction, the cytoplasmic calcium increase is by by PLC-P activated PI hydrolysis institute inductive, but ERK and Akt phosphorylation are that the kinase whose activation by MEK and P13 mediates respectively.Based on these results, the conformational change of the zygotic induction FPR of peptide part and FPR as if, conformational change causes acceptor and G-protein-mediation PLC-β or G-protein-mediation PI-3 kinases coupling.Because some peptides of the present invention can not induce cytoplasmic calcium to increase (WKGMVm, WKRMVm and D-Met6 substituted peptide), but can be through the pathway stimulation ERKs phosphorylation of PI-3 kinases-independently, as if peptide is attached to FPR and has induced conformational change, this conformational change is that the signal conduction of PI-3 kinase activator and MEK-mediation is needed, thereby causes the chemotactic migration of RBL-2H3 cell.

Existing reporting: two kinds of formyl peptide receptor family isoacceptors not, FPR and FPRL1 in congenital immune response, play a significant role (1,2).Up to now, several different parts sources comprise formyl peptides lipoxin A4, and existing report can combine (1) with FPR or FPRL1.Report such as Le WKYMVm can combine (12) with FPR and FPRL1.

In the present invention, the present inventor has confirmed with other amino-acid substitution Tyr ³Or D-Met ⁶To lose FPR and can not lose PLC-mediation cytoplasmic calcium-increases activity (Fig. 1) of FPRL1.This result shows Tyr ³And D-Met ⁶Be the key that activates PLC by FPR, but be not like this FPRL1.Part-the binding site that can derive FPR and FPRL1 from this result is different.

GPCRs comprise FPR heterogeneous by being attached to-trimerization G-protein and signal conduction in the inducing cell, the key amino acid residue (23,24) that relates to acceptor in the coupling of G-protein is attempted to disclose always by many study group.For FPR, Miettinen etc. have made up 35 kinds of sudden change FPRs and have investigated sudden change to the G-protein coupling of FPR and the influence of cell signaling.According to this article, S63, D71, R123 and C124/C126 are to the coupling of G-protein and FPR extremely important (24).In these mutant, the R123A mutant can not mediate the transfer of calcium, but can induce ERKs phosphorylation (24) by fMLF.Inferred Asp122 and Arg123, it has constituted conservative (D/E) RY structural domain (DRC among the FPR), has participated in the hydrogen bond network (24) of stable acceptor inactive form.In this was inferred, part was attached to the change that acceptor causes hydrogen bond network, and for example arginic exposure in the DRY structural domain of some amino-acid residue, thus can with G-protein interaction (24).Have been found that some peptides such as WKGMVm can induce the ERKs phosphorylation but can not cause that calcium shifts.Therefore judge that the receptor conformation whether WKYMVm or WKGMVm and combining of FPR induce difference changes extremely important; Although promptly WKYMVm can induce the conformational change of FPR, comprise the change of the hydrogen bond of DRY structural domain, WKGMVm has only caused that significant receptor conformation changes, and does not influence the DRY hydrogen bond.Under the situation of FPRL1, it also comprises DRY structural domain (DRC among the FPRL1), but does not investigate its effect in the G-albumen coupling.In these results, not only WKYMVm but also WKGMVm induce the calcium in the FPRL1-expression RBL cell to shift (Figure 1B).These results show that WKYMVm or WKGMVm do not influence the hydrogen bond of DRY structural domain among FPR or the FPRL1.Perhaps, other binding pocket participates in the peptide combination, and in following work, the residue of discerning in the difference binding pattern that relates to WKYMVm or WKGMVm and FPRs is extremely important.

Up to the present, reported that some native ligands can difference ground adjusting FPR.In immunity system, it is necessary that the difference of flailing action or chemotactic migration is regulated meticulousr adjusting.From this angle, identification is extremely important as the work that can regulate to difference the FPR part among the present invention.

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Sequence table

＜110〉POSCO (POSCO Co., Ltd.)

POSTECH Foundation

＜120〉immunomodulatory peptides

<130>OPP030092KR

<150>US60/352,930

<151>2002-01-29

<160>24

<170>Kopatentln 1.71

<210>1

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>1

Trp-Lys-Gly-Met-Val-Met

1 5

<210>2

<211>6

<212>PRT

＜213〉artificial sequence

<220>

Immunomodulatory peptides

<223>

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>2

Trp-Lys-Tyr-Met-Gly-Met

1 5

<210>3

<211>6

<212>PRT

＜213〉artificial sequence

<220>

Immunomodulatory peptides

<223>

<400>3

Trp-Lys-Tyr-Met-Val-Gly

1 5

<210>4

<211>6

<212>PRT

＜213〉artificial sequence

<220>

Immunomodulatory peptides

<223>

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>4

Trp-Arg-Tyr-Met-Val-Met

1 5

<210>5

<211>6

<212>PRT

<213>

Artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>5

Trp-Glu-Tyr-Met-Val-Met

1 5

<210>6

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>6

Trp-His-Tyr-Met-Val-Met

1 5

<210>7

<211>6

<212>PRT

＜213〉artificial sequence

<220>

Immunomodulatory peptides

<223>

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>7

Trp-Asp-Tyr-Met-Val-Met

1 5

<210>8

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>8

Trp-Lys-His-Met-Val-Met

1 5

<210>9

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>9

Trp-Lys-Glu-Met-Val-Met

1 5

<210>10

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>10

Trp-Lys-Trp-Met-Val-Met

1 5

<210>11

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>11

Trp-Lys-Arg-Met-Val-Met

1 5

<210>12

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>12

Trp-Lys-Asp-Met-Val-Met

1 5

<210>13

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

＜221〉MOD_RES (modification residue)

<222>(6)

<223>D-Met

<400>13

Trp-Lys-Phe-Met-Val-Met

1 5

<210>14

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

<221>MOD_RES

<222>(6)

<223>D-Met

<400>14

Trp-Lys-Tyr-Met-Tyr-Met

1 5

<210>15

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

<221>MOD_RES

<222>(6)

<223>D-Met

<400>15

Trp-Lys-Tyr-Met-(Phe/Trp)-Met-

1 5

<210>16

<211>6

<212>PRT

＜213〉artificial sequence

<220>

Immunomodulatory peptides

<223>

<400>16

Trp-Lys-Tyr-Met-Val-Glu

1 5

<210>17

<211>6

<212>PRT

＜213〉artificial sequence

<220>

Immunomodulatory peptides

<223>

<400>17

Trp-Lys-Tyr-Met-Val-Val

1 5

<210>18

<211>6

<212>PRT

＜213〉artificial sequence

<220>

Immunomodulatory peptides

<223>

<400>18

Trp-Lys-Tyr-Met-Val-Arg

1 5

<210>19

<211>6

<212>PRT

＜213〉artificial sequence

<220>

Immunomodulatory peptides

<223>

<400>19

Trp-Lys-Tyr-Met-Val-Trp

1 5

<210>20

<211>5

<212>PRT

<213>A

<220>

Immunomodulatory peptides

<223>

<400>20

Trp-Lys-Tyr-Met-Val

1 5

<210>21

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

<221>MOD_RES

<222>(5)

<223>D-Met

<400>21

Lys-Tyr-Met-Val-Met

1 5

<210>22

<211>4

<212>PRT

＜213〉artificial sequence

<220>

Immunomodulatory peptides

<223>

<400>22

Lys-Tyr-Met-Val

1

<210>23

<211>4

<212>PRT

＜213〉artificial sequence

<220>

Immunomodulatory peptides

<223>

<220>

<221>MOD_RES

<222>(4)

<223>D-Met

<400>23

Tyr-Met-Val-Met

1 5

<210>24

<211>3

<212>PRT

＜213〉artificial sequence

<220>

＜223〉immunomodulatory peptides

<220>

<221>MOD_RES

<222>(3)

<223>D-Met

<400>24

Met-Val-Met

1

Claims (13)

1. peptide of forming by the aminoacid sequence that is selected from SEQ ID NO:4～SEQ ID NO:7.

2. the peptide of claim 1, wherein said peptide are isolating and are pure forms.

3. a pharmaceutical composition comprises that aminoacid sequence is selected from the peptide of SEQ ID NO:4～SEQ ID NO:7.

Among the claim 1-2 each described peptide or the described pharmaceutical composition of claim 3 preparation treatment by change that quantity of leucocyte or state of activation are followed or the medicine of the disease that causes in purposes.

5. the described purposes of claim 4, wherein disease is bacterium, mycoplasma, yeast, fungi or virus infection.

6. the described purposes of claim 5, wherein disease is an inflammation.

7. each described peptide or the described pharmaceutical composition of claim 3 increase among the host leukocyte count or promote the leukocyte activation state with the purposes in the medicine of treatment disease in preparation among the claim 1-2.

8. each described peptide or the described pharmaceutical composition of claim 3 induce the white corpuscle extracellular Ca2 to increase with the purposes in the medicine of treatment disease in preparation among the claim 1-2.

Among the 9 claim 1-2 each described peptide or the described pharmaceutical composition of claim 3 preparation induce by person monocytic cell or neutrophil super-oxide produce with the medicine of treatment disease in purposes.

10. each described peptide or the described pharmaceutical composition of claim 3 are induced the chemotactic migration with the purposes in the medicine of treatment disease in preparation among the claim 1-2 by the human peripheral blood mononuclear cell.

11. each described peptide or the described pharmaceutical composition of claim 3 induce flailing action in formyl peptide receptor express cell or class formyl peptide receptor 1 express cell with the purposes in the medicine of treatment disease in preparation among the claim 1-2.

12. an isolating Nucleotide, its base sequence of aminoacid sequence that is selected from the peptide of SEQ ID NO:4～SEQ ID NO:7 sequence by coding is formed.

13. comprise the carrier of claim 12 Nucleotide.

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